The European Histamine Research Society
41st Annual Meeting, May 2–5, 2012
Belfast, Northern Ireland
Editor: G. Sturman
In cooperation with:
K. Barrett (San Diego)
D. Bell (Belfast)
P.L. Chazot (Durham)
M. Ennis (Belfast)
B. Gibbs (Kent)
G. Lees (Dunedin)
F.L. Pearce (London)
This supplement was not sponsored by outside commercial interests. It was funded entirely by the publisher.
Meeting report of the European Histamine Research Society
G. Sturman
Last year’s meeting in Sochi, Russia was the most eastern meeting we have ever held whilst this year’s meeting (the 41st meeting of the European Histamine Research Society) was the most westerly one. We met in Belfast, Northern Ireland at the kind invitation of Professor Madeleine Ennis of Queen’s University Belfast. This was the second joint meeting with COST Action 0806—see following article. These meetings took place in the Hilton Hotel, Belfast (2nd–5th May 2012). This was the first time we have ever met in Northern Ireland and we were situated right in the centre of Belfast beside the River Lagan and not far from the famous Harland and Wolff shipyards and the newly opened Titanic Exhibition Museum.
This year over 130 delegates attended representing 29 countries (most from the European countries, especially the UK but also from North and South America, Middle East, Australia and Japan). Some of the regular EHRS attendees could not attend and they were missed but a big welcome was given to all the new visitors, who we hope will return to future meetings. Most delegates arrived during the Wednesday. As usual the Council held their meeting late afternoon. Then there was a meeting for the COST BM 0806 WG 1 before our Welcome Reception; a delightful buffet with plenty of wine and fruit juices to drink. Old friends were greeted again whilst new ones were made.
On the Thursday, we began the meeting with the Opening Ceremony. We started with an introduction from our host Madeleine Ennis who informed us that ‘There were no strangers present—just friends we have not yet met!’ Then the Pro-Vice Chancellor for Research and Postgraduates at Queen’s University Belfast, Professor James McElnay welcomed us to QUB and Northern Ire-land. He spoke about the beginning of QUB in 1845 and gave an overview of the history and facilities within this University. Then our president, Anita Sydbom gave her welcoming talk, which centred round Irish folk customs, especially the folk dances and costumes. She then outlined the programme of this year’s meeting. She then presented the student bursaries; certificates and cheques (each for €500) to nine student members. The society is very grateful to the El-Sayed Assem family for sponsoring 2 student bursaries while the G.B. West Memorial Trust gave another and the remaining six were from our society. This was followed by the presentation of certificates to 16 COST BM0806 Early Stage Cost researchers. Then there
was the presentation of Honorary Membership to two of our very special members; first to Professor Zsuzsanna Huszti (Budapest, Hungary) and then to Professor Helmut Haas (Dusseldorf, Germany). Anita gave the laudation to Zsuzsanna and Patrizio Blandina (Italy) to Helmut. Both Zsuzsanna Huszti and Helmut Haas have made significant contributions to our society in many different ways over the years. They were presented with a certificate beauti-fully written in Latin and sporting the society’s official seal. Then we heard the first of our plenary lectures. This was given by Holger Stark (Germany) and was entitled ‘Histamine H4 receptor—ligand, binding and activation’.
Then at the start of our coffee break, we had a group photograph taken. This was followed by the Walter Schu-nack Memorial Symposium in which Holger Stark started by giving an appreciation to Walter. This was followed by 4 oral presentations on the chemical aspects of histamine receptor ligands. All the presenters gave very positive references to Walter, his work and support and also to his wonderful sense of humour. Continuing our tradition, each oral session at this meeting was chaired by an experience scientist as well as a young EHRS member so that they could get experience of chairing oral sessions.
Then we quickly moved outside to get the coaches for our outing. We drove along the northern side of the Belfast Lough to Carrickfergus Castle, where we stopped to explore this 800 year old castle. Participants could be seen climbing up on the battlements and visiting the various castle rooms. Then we continued along the Causeway Coastal route which hugs the narrow strip of coastline between the sea and the high cliffs. We passed through some picturesque towns and villages and saw some spec-tacular scenery, even sighting Scotland on the horizon as well as Raithin Island. Finally we reached Northern Ire-land’s most famous World Heritage Site, the Giant’s Causeway, which was formed over 60 million years ago, when molten lava cooled suddenly on contact with water producing stone ‘benzene ring’ columns! Most people made their way down to actually clamber over the Giant’s Causeway. Then after warming ourselves up with hot drinks we continued inland to Ballymena and the Galgorm Hotel. Here we were made very welcome and had a won-derful evening meal in this spectacular manor house. During our dinner, we were entertained by 2 talented Irish musicians from the band Pure Blarney singing and playing
the guitar, tin whistle and bagpipes. We all agreed that it was an excellent end to the first day of our meeting.
Friday began with the G.B. West lecture which was given by Peter Bradding (Leicester University, UK) and was entitled ‘Interactions between mast cells and structural airway cells in the pathogenesis of asthma’. This was fol-lowed by poster sessions on ‘Histamine and the Nervous System’ and ‘Mast cells, Metabolism and Chemistry’. Then we listened to three lectures on ‘Novel highlights on mast cell functions’ (COST BM 1007). After lunch, like last year, we held a Round Table which was entitled ‘What’s new in H4research? (COST BM 0806). Robin Thurmond
(USA) lead a panel of histaminologists which included Elena Rivera (Argentina), Paul Chazot (UK), Ralph Gutzmer (Germany) and Hoger Stark (Germany) and needless to say this resulted in lively discussions. After the afternoon break, there was an oral session on ‘Mast cells and Inflammation’ followed by another one on ‘Clinical aspects of the H4 receptor’ (COST BM0806). Then the
WG3 COST Action BM0806 took place. After our evening meal, we took part in some Irish Whiskey Tasting. We were informed that whiskey was the ‘Water of Life’ and that Irish whiskey differed from Scotch as it was distilled three times rather than just twice. This thus gives Irish whiskey a much smoother taste. As predicted, there was much fun involved with this activity.
Our final plenary lecture by Beatrice Passini (Florence, Italy) started our Saturday’s meeting. Her lecture was entitled ‘H4R and neuroinflammation: insights from mouse
experimental autoimmune encephalomyelitis’ and she gave a very interesting lecture despite the projector not showing the true colours of her histopathology results. This lecture was followed by an oral session on ‘Histamine and the Nervous System’. After our coffee break, we viewed the poster presentations on ‘Histamine—H4receptor’ and also
‘Clinical aspects and cellular studies’.
Throughout the meeting the poster committee had been working very hard and as usual had a difficult task in identifying winning posters for the poster competition. Eventually first prize was given to F. Jantzen et al. from Hannover, Germany, second to S. Micallef et al., from Dublin, Ireland and third prize to L. Kay et al., from Sheffield, UK.
The final oral session of our meeting was to listen to our younger members (PhD students or not more than 3 year’s post-doctoral research) give their presentations for the EHRS Young Investigators Award. Regrettably one student had withdrawn at the last minute. It was another difficult task for the judges in differentiating between these four excellent presentations. The winner was Gustavo Provensi (Florence, Italy) with Naresha
Saligrama (Vermont, USA) and Tomas Perecko (Brati-slava, Slovakia) sharing joint second place and Yan Zhao (Lyon, France) being highly commended. To summarise the COST Action BM0806 work, Katherine Tiligada (Athens, Greece) told us about the ‘Progress and Limita-tions in H4R research.’ Then we held our General
Assembly. According to our statutes, a new Council had to be elected and after a ballot, Nick Carruthers (USA), Jian-Sheng Lin (France), Beatrice Passani (Italy) and Astrid Sasse (Ireland) were duly elected to serve on our Council. Additionally Anita’s second and last term of office as President has now finished. Paul Chazot (Dur-ham, UK) was unanimously elected as our new President. Many thanks were given to Anita for all her hard work to the society over the last 6 years. Our meeting ended with its traditional excellent Farewell Dinner followed by our award ceremony. The certificates and prizes were given out. Then as usual we had our singing session, beginning with ‘‘Anita’s Thank You Song’’ (sung to the tune of ‘It’s a long way to Tipperary’) as a big thank you to Madeleine and her team for the excellent meeting, and then we sung our EHRS Anthem. Afterwards ‘Haste to the Wedding’ played for us and we had a go at some Irish Ceilidh dancing—great fun was had by all.
We all agreed that the Northern Irish team, especially Madeleine had hosted a very well organised meeting with excellent science. Our thanks were given to all of them. The next meeting will be held in Lodz, Poland (8–11 May, 2013) at the kind invitation of Agnieszka Fogel.
Participants of the 2012 meeting of the EHRS
Department of Pharmacology and Therapeutics, School of Biomedical and Health Sciences, Kings College London, London, SE1 1UL, UK. E-mail: gill.sturman@virgin.net
Honorary Memberships in the European Histamine Research Society
P. Blandina, A. Sydbom, H. Haas, G. Sturman
There are three types of membership in the EHRS: ordin-ary, corporate and affiliated, and honorary (life). The highest award is that of honorary membership which is only given to very special people. To obtain honorary membership, the person has to be elected by over two-thirds of the ordinary members at the General Assembly. There are only 10 current Honorary Members of the Society; Madeleine Ennis, Robin Ganellin, Wilfried Lorenz, Piero Mannaioni, Bruno Mondovi, Fred Pearce, Henk Timmerman, Ingrid Olhagen-Uvna¨s, Takehiko Watanabe and Jean West. Our former Honorary Members include Sir James Black, Franc Erjavec, Jack Peter Green, Czeslaw Maslinski, Wolfgang Schmutzler, Walter Schunack, Bo¨rje Uvna¨s and Geoff West. At this meeting, the society awarded Honorary Membership to two special people who have both contributed significantly to the EHRS; they are Professor Zsuzsanna Husti and Professor Helmut Haas.
The oration for Professor Zsuzsanna Husti was given by our President, Anita Sydbom. Zsuzsanna Huszti, of the famous Semmelweis University in Budapest, Hungary, was born on the 16th of March 1936 in Szeged. In 1954 she entered the University of Szeged in the faculty of Natural Sciences where she studied chemistry. After getting her diploma in chemistry 1959, she worked as a research assistant at the Central Research Laboratory of the Szent-Gyo¨rgyi Albert Medical School in Szeged. In 1962, Zsuzsanna started her doctorate work in biochemistry at the Eo¨tvo¨s Jo´zsef University, Budapest. At that time she also had a position as clinical chemist in the Central Hospital, Gyula, and as a research assistant at the Department of Pharmacology in the Drug Research Institute, Budapest. From 1971 to 1981 she was head of the Radioactive Lab-oratory at the Department of Biochemistry, Drug Research Institute, Budapest. From 1997 she became the head of the Neurobiobiology Unit in the Department of Pharmacody-namics, Semmelweis University of Medicine, Budapest.
Although her first publication was in 1963, her first one on histamine was in 1967 and her Ph.D. thesis (1972) was entitled ‘‘Heterogeneity of monoamine oxidase in rat brain’’. This was followed by postdoctoral position in the Department of Neurobiology, at McGill University, Mon-treal, Canada working with Professor Theodore L. Sourkes. This resulted in a fruitful collaboration and many papers about histidine metabolism. In 1994 Zsuzsanna produced a
second thesis; this time for Doctor of Biological Science degree. This thesis was entitled ‘‘Histamine in the nervous system: regulation of histamine synthesis and function in the brain’’. She has published over 75 publications; most are concerned with histamine. She is/was a member of several scientific societies, namely the Hungarian Society of Biochemistry, International Society for Neurochemistry (ISN), Hungarian Pharmacological Society, European Society for Neurochemistry (ESN), International Brain Research (IBRO) and New York Academy of Sciences plus our society.
Zsuszanna Huszti has had a long standing connection with the EHRS. She was invited by Professor Maslinski, who had read her articles, to the very first informal meeting of the ‘Histamine Club’ held in Lodz, Poland 1971. After this meeting she has visited 36 EHRS meetings in total. She was the EHRS National Secretary for Hungary from 1972 to 2000. She gave an EHRS Invited Lecturer at the meeting in Eger (2004) with the title: Histamine uptake by non-neuronal cells of the brain: properties and function. She brilliantly organized two unforgettable EHRS meet-ings in Hungary, the 9th EHRS Meeting 1980 in Visegrad and the 23rd EHRS Meeting 1994 in Budapest. Many pictures from the different EHRS meeting can be seen under Previous Meetings on our website http://www. EHRS.org.uk. Zsuzsanna Huszti has for many years been devoted to our society, interested in science and young researchers and always is putting interesting questions to the speakers at our meetings. So with this it is a great honour to present the EHRS Honourary Membership to Zsuzsanna Huszti.
The oration for Professor Helmut Haas was given by Professor Patrizio Blandina. Helmut grew up in the south-west corner of Germany. After naval military service he studied medicine in Basel, Switzerland and Freiburg, Ger-many, where he obtained his M.D. (1968) for a dissertation on aphasia. Then he worked in the Psychiatric and Neuro-logical University—Clinics in Basel before taking a postdoctoral position in the Department of Pharmacology, Cambridge, England. This was followed by a number of years as head of the Neurophysiology-Laboratory in the Neurosurgery-Department Zu¨rich and visiting Professor-ships on sleep research at Harvard-University in 1986 and 1989. In 1987 Helmut became Professor and Head of the Department of Biophysics at
Johannes-Gutenberg-Universita¨t Mainz, Germany; in 1991 he took the Chair of Neurophysiology at Heinrich-Heine-Universita¨t Du¨sseldorf. In 1971 Helmut began to work on histamine in the brainstem. He has since devoted much of his scientific life to this fascinating field characterizing histamine actions in the brain (e.g. Nature 1975, 1983, Neuron 1993) and later the properties of histaminergic neurons in the tubero-mamillary nucleus using mainly electrophysiological methods. He has published over 200 peer-reviewed papers and serves on the editorial boards of several journals. His research was financed by the National Institutions in Switzerland and Germany and the Human Frontiers Sci-ence Programme (network mechanisms of memory trace formation) as well as the European Community (Consor-tium ‘‘food, mood and sleep’’ on orexins). Helmut, besides being a regular attendee of our meetings, hosted the 33rd EHRS meeting in 2004 near Du¨sseldorf. He is now, retired but not tired, cooperating with his wife (Prof. Olga Sergeeva) on new challenges of the histaminergic system in the brain. Helmut truly deserves the accolade of being elected an Honorary Member of this society.
Helmut Haas, Anita Sydbom (President) amd Zsuzsanna Huszti at the Farewell Dinner
Department of Pharmacology and Therapeutics, School of Biomedical and Health Sciences, Kings College London, London, SE1 1UL, UK. E-mail: gill.sturman@virgin.net
Meeting Report of the Cost Action BM0806 ‘Recent advances in histamine
receptor H
4R research’
E. Tiligada
The main 2012 meeting of COST Action BM0806 ‘Recent advances in histamine receptor H4R research’ was hosted
by Madeleine Ennis (Secretary of the Action, The Queen’s University of Belfast, UK) and took place jointly with the 41st Annual EHRS conference held in the Hilton Belfast hotel, UK. The key objective of the Action BM0806 is to foster a multidisciplinary approach to histamine H4
receptor (H4R) research and to focus on the current state of
play pertaining to the basic understanding and the huge therapeutic potential of this new drug target. The Action builds up strong European interdisciplinary links among more than 150 senior and young scientists from at least 40 research groups based on 20 COST (AT, CH, CZ, DE, DK, EL, ES, FI, FR, HU, IE, IL, IT, LT, NL, PL, SE, SI, SK, UK) and 4 non-COST (AR, NZ, US, RU) countries, who actively participate in the 4 mutually interlocking working Groups (WG) of the Action.
H4R is the most recently identified histamine receptor
subtype, which is primarily linked to immunomodulation and to a variety of inflammatory disorders. The develop-ment and the evaluation of selective H4R ligands aim to
promote the translational perspective in this field of research. COST Action BM0806 has a firm foundation for managing research into the H4R function and therapeutic
potential due to the combined efforts of scientists from multiple disciplines including biochemistry, molecular and cellular biology, genetics, chemistry, bioinformatics, phar-macology and medicine. Members of the COST Action are dominating the publications in this area, thus making Eur-ope the leader in the field. This joint conference enhanced the previously established links with the EHRS, while the rapid and productive result dissemination and the direct exchange of ideas will enable the expert teams to work more efficiently towards the potential exploitation of this new drug target. Additionally, a delegate from Japan, the major non-COST country where H4R research outside BM0806 is
pursued, applied to join the Action. Importantly, the train-ing of Early Stage Researchers (ESRs) in all aspects of H4R
research was broadened and their mobility by using the Short-Term Scientific Mission (STSM) COST tool was stimulated and encouraged during this joint activity.
The COST conference attracted 58 Action members from 22 member countries and comprised 3 COST Plenary Lectures, 2 COST sessions with 13 oral presentations and
14 peer-reviewed posters (5 related to STSMs), a round table session, a guest symposium from members of the COST Action BM1007, which concentrates on mast cell and basophil function, a BM0806 Management Committee meeting and WG1, 2, 3 & 4 meetings. In keeping with the tradition started at the Durham meeting in 2010, symposia and poster sessions were nearly all co-chaired by ESRs together with senior scientists. Sixteen ESRs received awards for their contribution to H4R research, while the
winner and the 2nd prize winner in the poster competition of this Joint meeting were both Action member ESRs (Franziska Jantzen, Hannover Medical School, DE; Stephanie Micallef, Trinity College Dublin, IE). In the Young Investigator Award, Toma´sˇ Perecˇko (Slovak Academy of Sciences, SK) received joint 2nd prize as a result of the work that he performed during a COST BM0806 STSM.
An elegant description of H4R ligands and the
approa-ches for lead development was provided by Holger Stark (Goethe University, Frankfurt, DE). The data on the char-acterization of the H4R binding pocket by molecular
dynamics simulations and the binding properties of H4R
agonists, inverse agonists and neutral antagonists will be of immense use to the members of the COST Action. New information was provided by Beatrice Passani (University of Florence, IT) on the involvement of the H4R in
neuro-inflammation and in particular in a mouse model of multiple sclerosis. Strategies aimed at interfering with the histamine axis may have relevance in the therapy of autoimmune diseases of the central nervous system (CNS) as histamine may determine a shift in T helper cell sub-population, influence migration of lymphocytes and myeloid cells during CNS invasion, interfere with antigen presentation at the immune synapse level and determine variations in normal neuronal functions. During the guest symposium of the COST Action BM1007, Uli Blank (In-serm U699, FR) proposed the late signalling events in mast cell degranulation as a novel target for the development of drugs to block mediator release; Gunnar Nilsson (Karo-linska Institutet, SE) reported on the secretion of large variety of oxylipins by mast cells and Francesca Levi-Schaffer (The Hebrew University of Jerusalem, IL) described the interactions between mast cells and eosino-phils, which serve to perpetuate the inflammatory response.
The following highlights of the COST Action BM0806 presentations show the advancements on H4R research.
Rob Thurmond (Johnson & Johnson, US) offered insights on the interaction between H4R and TLR activation in
vivo, which can drive inflammatory responses. Arianna Rosa (ESR, University of Turin, IT) suggested a novel use for H4R blockers in pulmonary fibrosis and also showed
that the H4R expressed in the kidney is up-regulated in
diabetic male rats. Toma´sˇ Perecˇko provided novel infor-mation on the presence of H4Rs in human neutrophils and
on their involvement in the inhibition of neutrophil degranulation. Interestingly, Maristella Adami (University of Parma, IT) reported on the effective inhibition of car-rageenan-induced oedema by H4R agonists rather than
antagonists. Spyridon Chalkiadakis (ESR, University of Athens Medical School, EL) provided further evidence on the putative constitutive H4R-mediated automodulatory
histamine function in the rat conjunctiva using novel 2,4-diaminopyrimidine derivatives targeting the receptor. The presence of H4Rs on peptidergic Ad fibres (CGRP) and
C-fibres (substance P) reported by Mwape Katebe (ESR, University of Durham, UK) could have implications not only in chronic pain but also in chronic cough, while evi-dence provided by Diego Martinel Lamas (ESR, University of Buenos Aires, AR) pointed to the potential therapeutic use for H4R agonists in the treatment of breast cancer.
Novel approaches to H4R research were presented by
Gniewomir Latacz (ESR, Collegium Medicum Jagiellonian University, PL) who found that some N-methylpiperazine derivatives influenced the motility of the promoter of PTEN, one of the most frequently mutated tumor
suppressor genes in human cancer as well as by Anwar Rayan (GeneArrest, IL) who described the methods used to alter H4R gene expression by novel and highly specific
molecules which bind tightly and selectively to predeter-mined sequences in the major groove of double stranded DNA. Hubert Schwelberger (Medical University Inns-bruck, AT) provided information on the progress of the histamine methods and tools database (HMTD), which will be of immense use to all working in the field. Finally, the Action Chair Katerina Tiligada (University of Athens Medical School, EL) presented an overall update on current H4R research, emphasising that a large body of evidence
identifies the H4R as a central player in initiating and
propagating immune responses, yet the variability in H4
R-mediated signals and in the pharmacological properties of H4R ligands has to be carefully considered when
charac-terizing H4R functions in in vivo models of disease and
translating preclinical data to clinical human settings. Overall, this joint COST Action BM0806/EHRS con-ference attracted all the major players who have interest in H4R-related research and drug development and it was
characterised by intense and fruitful discussions between delegates, which are expected to meet the major challenges in understanding the functional properties and the thera-peutic potential of the H4R.
E. Tiligada
Department of Pharmacology, Medical School, University of Athens, 11527 Athens, Greece.
PROGRESS AND LIMITATIONS IN H4R
RESEARCH E. Tiligada
Histamine elicits pleiotropic actions largely through binding to four currently known G protein-coupled receptors (GPCRs), designated as H1R–H4R. The latest concept that
histamine exerts immunomodulatory actions in inflamma-tion through H4R signalling and the potential exploitation of
this activity for a range of poorly treatable chronic inflam-matory diseases are currently under worldwide evaluation. Although the large body of evidence identifies the H4R as a
central player in initiating and propagating immune responses, the cell and tissue variability in H4R-mediated
signals and the profound intra- and inter-species differences in potency, selectivity and off-target effects of H4R ligands
hamper investigations and call for more cautious interpre-tation of H4R-mediated effects in vivo. For instance, the
complex pharmacology of H4R ligands can be partly
attrib-uted to the functional selectivity exhibited by many GPCRs under different experimental or physiological environments. In this multifaceted system of immunoregulation further experimental approaches are needed to address numerous unresolved questions. For instance, what is the contribution of the H4R in differentiating the phenotypes and chemotaxis
of immunocompetent cells in inflammation? What are the molecular mechanisms underpinning H4R cross-talk with
immune-relevant pathways, such as TLR signalling and TH1/
TH2 polarisation, and how would these interactions be useful
in identifying more effective therapeutic targets for inflam-matory diseases? Finally, it would be interesting to know whether autocrine or paracrine mechanisms are in operation considering that mast cells, the main histamine source in the body, express histamine receptors. Establishing the benefi-cial end-points of the histamine-mediated orchestration of the complex immune response is an altruistic challenge of EU RTD FP7 COST Action BM0806: Recent advances in histamine receptor H4R research.
Department of Pharmacology, University of Athens Medical School, 11527 Athens, Greece. E-mail: aityliga@ med.uoa.gr
H4R AND NEUROINFLAMMATION: INSIGHTS
FROM MOUSE EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS
M.B. Passani
The histaminergic system has been postulated to have a role in the pathogenesis of autoimmune diseases and there
are several lines of evidence suggesting a key regulatory role of histamine in the widely used murine model, experimental autoimmune encephalomyelitis (EAE). Therefore, strategies aimed at interfering with the hista-mine axis may have relevance in the therapy of CNS autoimmune disease. Given the distribution of the hista-mine H4 receptor (H4R) on immune cells and its primary role in inflammatory functions it has become a very attractive target for the treatment of asthma and autoim-mune diseases. Furthermore, recent evidence has shown the topological and functional localisation of the H4R in the CNS of both humans and rodents. However, the picture is more complex than expected, as recent data showed that the activation, and not the antagonism, of the H4R leads to reduced pro-inflammatory capacity of a subpopulation of dendritic cells found in inflamed tissues in atopic derma-titis. Results from our laboratory are also pointing in this direction, as H4R antagonists tested in EAE murine model appear to exacerbate several parameters of the disease and up-regulate H4R expression on T and dendritic cells in the spinal cord of EAE mice. Accordingly, H4R KO mice develop more severe EAE.
The complex and apparently controversial results sum-marized here, on the one hand provide compelling evidence that the H4R has multiple roles in the function of various cellular elements that may be associated with immune disorders. However, these observations challenge the scientific community to develop the adequate treat-ments for these pathological conditions.
Dipartimento di Farmacologia Preclinica e Clinica 50139 Firenze, Italy. E-mail: beatrice.passani@unifi.it
BEHAVIOURAL ANALYSIS OF H4RECEPTOR
KNOCKOUT MICE
K. Rossbach, M. Bankstahl, W. Ba¨umer
Histamine is a well known neurotransmitter that is involved in the regulation of many different brain functions such as waking-sleep cycle, anxiety, cognition, learning and memory as well as basic homeostatic functions. All four histamine receptors (H1-4R) are located in the brain.
The expression of the H4R in the human and rodent brain
has only recently been reported and its functional role remains to be elucidated. In this study a first set of behavioural tests was performed twice with H4R knockout
mice in comparison to age-and sex-matched wild-type mice (BALB/c) (4 male and 4 female mice per group). For a general behavioural screen, a subset of tests from the Irwin screen was performed (7 males and 10 females/ group). General physical characteristics or sensorimotor
reflexes showed no obvious abnormalities in the group of H4R knockout mice. However, H4R knockout showed an
enhanced escape response to approach and struggled more when touched or restrained by tail. For analysing the general activity and anxiety-related behaviour, the open field test, the light and dark box and the elevated plus maze test were performed. Overall, the H4R knockout mice
showed only marginal differences in exploratory or anxi-ety-like behaviour. Furthermore, spatial learning and memory were tested in the Morris water maze test (10 females/group). The H4R knockout showed an apparent
improved performance in the Morris water maze test compared to the poor performance of the wild-type mice in this experimental setting. In summary, the H4R knockout
mice seem to react with hyperactivity to handling, but showed no gross differences in other test parameters of the Irwin screen or in their exploratory or anxiety-like behav-iour. The apparent improved performance in the Morris water maze test of the H4R knockout mice may be a first
hint for a better learning and memory behaviour of these mice. Thus, the present study reveals that there are dif-ferences in the behavioural phenotype of the H4R knockout
mice, but further studies are required to confirm these differences and in more detail.
Supported by COST Action BM0806
Institute of Pharmacology, Toxicology and Pharmacy, University of Veterinary Medicine Hannover, 30559 Han-nover, Germany. E-mail: kristine.rossbach@tiho-hannover.de
SPECIFIC HISTAMINE H4RECEPTOR
ANTAGONISTS ACT AS POTENT MODULATORS OF MAMMAL VESTIBULAR FUNCTION
E. Wersinger, G. Desmadryl, S. Gaboyard-Niay, A. Brugeaud, J. Dyhrfjeld-Johnsen, C. Chabbert
Histamine is a naturally occurring biological amine that exerts a range of effects on many physiological processes through the activation of four different G protein-coupled histamine receptors (H1R-H4R). Besides their localization in the immune system, H1-H4 receptors are also found in the central and peripheral nervous systems. Recent work has shown H3R expression in mammalian vestibular neu-rons and has shown that their antagonist, betahistine, routinely used in the treatment of vertigo can act periph-erally by inhibiting the afferent discharge recorded from the vestibular nerve in several species.
We aimed to further explore the expression and role of H4R in Long Evans rat vestibular system by combining RT-PCR and immunohistochemistry experiments, as well
as pharmacological and behavioral tests. Using this mul-tidisciplinary approach we first confirmed the expression of H1 and H3 receptors and further demonstrated the locali-zation of H4R in primary vestibular neurons cytoplasmic membrane. In vitro, whole cell patch-clamp recordings from P4 to P7 cultured vestibular neurons revealed strong and reversible inhibitory effects on evoked action potential firing by H4R antagonists JNJ10191584 and JNJ7777120 with IC50 in the lmolar range (n [ 5 cells/concentration
tested). In vivo, the effect of these antagonists on experi-mentally induced severe vestibular deficits were evaluated in three different animal models using a rating system based on multiple criteria: circling, head bobbing and head tilt, tail hanging and air righting reflexes. Each H4R antagonist significantly alleviated induced vestibular defi-cits by 20–30 % (n [ 8 animals for control, sham and treated conditions). By contrast, neither of the H3 receptor antagonists tested as reference compounds had significant effects.
This study demonstrates the role of H4R antagonists in modulating vestibular function and suggests that they are strong candidates for a novel, highly efficient treatment of vertigo crisis caused by peripheral vestibular dysfunction. SENSORION pharmaceuticals, Institute for Neurosci-ences of Montpellier, 34090 Montpellier, France. E-mail: eric.wersinger@sensorion-pharma.com
DIFFERENTIAL CHANGES IN H4R EXPRESSION
IN ACUTE AND CHRONIC INFLAMMATORY PAIN MODELS
M. Katebe, N. Lethbridge, P.L. Chazot
The histamine H4receptor is a G-protein coupled receptor
that is primarily expressed on cells involves in inflamma-tion and immune responses such as eosinophils, mast cells, dendritic cells and T-cells. It has been postulated to have immunomodulatory functions in these cells such as acti-vation, migration and production of chemokines and cytokines.
Immunohistochemical studies from our laboratory and others have revealed the expression of the receptor on sensory neurons, small and medium sized diameter neurons and lamina I and II of the spinal cord suggesting a potential new target for the modulation of pain. The H4R antagonist
JNJ7777120, exhibits analgesic effects in Complete Fre-und’s adjuvant (CFA) pain models.
The aim of the present study was to investigate the changes in H4receptor expression that occur in the acute
(24 h) and chronic (16 days) phases of Complete Freund’s adjuvant induced inflammation as compared to non-treated
animals (in collaboration with GSK (UK) and Pfizer (UK), respectively). Our hypothesis was that H4receptor protein
would be differentially altered during the acute and chronic phases of inflammatory-induced pain.
Using our well validated anti-hH4receptor antibody which
cross-reacts with rodent tissue, western blot analysis of skin and spinal cord tissue samples obtained from CFA-induced male Wistar rats, acute (n = 10) and chronic (n = 4) inflammatory pain models showed an apparent upregulation in the expression of H4receptors at the skin but not the spinal
cord. In contrast, this apparent up-regulation was not observed later in the chronic phase in the skin, and an apparent down-regulation of expression was seen in the spinal cord. We are currently formally identifying the cell types where these changes are occurring, namely immune and/or neuronal. This indicates both topological and temporal differences in H4R
expression occur in inflammatory pain states
School of Biological & Biomedical Sciences, Durham University, DH1 3LE. Durham, UK. E-mail: m.a.katebe@ durham.ac.uk
ROLE OF HISTAMINE H4R IN
BLEOMYCIN-INDUCED PULMONARY FIBROSIS
A. Pini, D. Bani, M.B. Passani, R.L. Thurmond, H. Stark, E. Masini
Pulmonary fibrosis is a progressive and lethal illness characterized by inflammation and abnormal remodeling of lung parenchyma. No cure exists for this disease. There are various models used for the study of pulmonary fibrosis, among these, the bleomycin one is the best characterized murine model in use. Bleomycin alters oxidant/antioxidant balance and ROS overproduction activates several intra-cellular signaling pathways, leading to pro-inflammatory cytokine production. The histamine H4R, expressed on cell
of immune origin, plays an important role in inflammatory process. We previously demonstrated that JNJ7777120 (JNJ), a selective H4R antagonist, potentiates the beneficial
effect of naproxen in this experimental model. The aim of the present study was to investigate the role of different H4R ligands in controlling inflammation and pulmonary
fibrotic process induced by bleomycin. C57/bl6 mice were treated with vehicle, JNJ (total dose 40 mg per Kg/bw) or ST-1124 (partial inverse agonist), ST-1006 (partial agonist) and ST-994 (neutral antagonist) at equimolar doses, released by micro-osmotic pumps for 21 days. Airway resistance to inflation, an index of lung stiffness, was assayed and lung tissue processed to evaluate inflammation and fibrosis. Our results indicate that JNJ and ST-994 exert an anti-inflammatory effect, as shown by the significant
decrease in the levels of PGE2, MPO, an index of leukocyte
infiltration, and TBARS, markers of oxidative stress. They also reduce the relative number of goblet cells, the thick-ness of smooth muscle layer (parameters of inflammation-induced adverse bronchial remodeling), the level of pro-fibrotic cytokine (TGF-b) and collagen deposition; these effects are accompanied by a decrease in airway resistance to inflation. The compounds ST-1112, partial inverse agonist and ST-1006, partial agonist, exerted a modest effect on reducing inflammation and preventing the fibrotic process.
Taken together our results indicated that H4R blockade
is associated with an anti-inflammatory and anti-fibrotic effect and may offer a new therapeutic option for the treatment of Th2-dependent lung inflammatory disease.
Supported by COST Action BM0806.
Department of Preclinical and Clinical Pharmacology, University of Florence, Viale G. Pieraccini n.6, 50139 Florence, Italy. E-mail: emanuela.masini@unifi.it
ANTAGONISM OF THE HISTAMINE H4
RECEPTOR REDUCES LPS-INDUCED TNF PRODUCTION IN VIVO
J.M. Cowden, F. Yu, M. Challapalli, J.-F. Huang, W.-P. Fung-Leung, J.Y. Ma, J.P. Riley, M. Zhang, P.J. Dunford, R.L. Thurmond
Antagonism of the histamine H4receptor (H4R) has been
shown to be anti-inflammatory in a number of preclinical disease models, however the exact mechanisms behind this are still being uncovered. In vitro, the receptor has been shown to interact with TLR and impact inflammatory mediator production from a number of different cells types. Here it is shown that this interaction can also occur in vivo. Two different H4R antagonists, JNJ 7777120 and JNJ
28307474, inhibited LPS-induced TNF production in mice. Furthermore, this production was also reduced in H4
R-deficient mice. TNF mRNA analysis showed that the major source of the cytokine was the liver and not blood, and that the H4R antagonist only reduced the expression levels in
the liver. The inhibition was only observed with in vivo administration of both the H4R antagonists and LPS
sug-gesting that the effect is mediated by tissue resident cells. In support of this conclusion, depletion or inactivation of macrophages reduced the TNF levels and eliminated the H4R sensitivity. Treatment with an H4R antagonist also
reduced induced liver injury and blocked LPS-enhanced lung inflammation in mice. In conclusion, the data support an interaction between H4R and TLR
Janssen Research and Development, L.L.C. San Diego, CA 92121, USA. E-mail: rthurmon@its.jnj.com
THE HISTAMINE H4RECEPTOR BLUNTS
NEUTROPHIL DEGRANULATION
BY PREVENTING BETA 2 INTEGRIN-DEPENDENT SIGNALLING
T. Perecko, V. Brown, D. Comer, R. Thurmond, K. Dib, M. Ennis
Histamine is a biogenic amine implicated in allergic asthma. In the airways, its main targets are eosinophils, smooth muscle and the endothelium. There are 4 types of histamine receptors (H1R, H2R, H3R, and H4R). All
belong to the G protein-coupled receptor family. Hista-mine has also been shown to regulate the functions of other immune cells including neutrophils. However, little is known on the nature of the histamine receptors involved in the regulation of neutrophil functions. We investigated whether the H4R regulates beta2 integrin-dependent
adhesion and degranulation in human neutrophils. Neutrophils were isolated from blood by Dextran sedi-mentation and centrifugation through Ficoll-Hypaque. Neutrophils adherent to fibrinogen (a ligand for beta2 integrins) were stained with crystal violet and the OD of the eluted dye was read using a spectrophotometer. Degranulation was assessed by ELISA by measuring the release of lactoferrin. The chemoattractant fMLP (10-7M) induced a beta2 integrin-dependent adhesion and degran-ulation of human neutrophils. In contrast, histamine by itself did not have such an effect. However, histamine (10-6 M) blocked fMLP-induced adhesion-dependent degranulation of human neutrophils by ca. 60 %. The H4R
agonist JNJ28610244 (10-5 M) reduced release to ca. 38 % of fMLP alone. Moreover, the H4R antagonist
JNJ7777120 prevented the inhibitory effect of histamine on neutrophil degranulation. Interestingly, neither hista-mine nor the H4R agonist JNJ28610244 blocked
fMLP-induced beta2 integrin-dependent neutrophil adhesion indicating that histamine did not interfere with the expression or change of conformation of beta2 integrins induced by the chemoattractant fMLP. Our results dem-onstrate for the first time that the H4R blocks neutrophil’s
anti-microbial functions.
Supported by a STSM from COST Action BM0806. The work has been awarded a Young Investigators Award by the EHRS 2012.
Centre for Infection and Immunity, Queen’s University Belfast, Belfast, BT9 7BL, UK. E-mail: k.dib@qub.ac.uk; m.ennis@qub.ac.uk
H1VERSUS H4ANTI-HISTAMINES IN HUMAN
NEUTROPHIL OXIDATIVE BURST
R. Nosal, K. Drabikova, V. Jancinova, T. Perecko, A. Lojek, M. Ciz, J. Kralova
Professional phagocytes play a crucial role in phagocytosis, immune reactions and pathological processes, including inflammation. Histamine possesses a regulatory role in these reactions. We found that H1R anti-histamines (AH)
inhibited the oxidative burst of professional phagocytes according to their chemical structure and physico-chemical properties. In this study, we compared the effect of H1R
and H4R AH on the oxidative burst of human blood and
isolated neutrophils by means of luminol and/or isoluminol enhanced chemiluminescence (CL) in vitro. Thirteen compounds of the 1st and 2nd H1R AH generation, and
JNJ7777120, JNJ10191584 and thioperamine, H4R AH
were analysed. Most of the H1R AH tested
dose-depen-dently inhibited stimulated CL produced by neutrophils. Histamine was effective only at the extracellular level. H1R
AH inhibited CL both at extracellular (scavenging) and intracellular level of neutrophils (suggestive of interference with regulatory enzymes). It is expected that the inhibition is a result of nonreceptor rather than receptor interaction. The H4R AH JNJ7777120 and JNJ10191584 tested showed
very weak inhibition on whole blood and extracellular CL, without any dose–response relationship and they were not active intracellularly. Thioperamide potentiated CL both in whole blood and in isolated neutrophils. The difference between H1R and H4R AH effect on oxidative burst
requires further analysis at the molecular level and through structure–activity relationship. In comparison with H1R
AH, the H4R AH compounds tested displayed a very low
scavenging and intracellular antioxidative and oxidative burst suppressing activity.
Supported by projects VEGA 2/0003/07, APVV 0315-07 and 0052-10 and COST BM0806.
Institute of Experimental Pharmacology SAS, 88104 Bratislava, Slovakia. E-mail: exfarano@savba.sk
EFFECTS OF HISTAMINE H4RECEPTOR
AGONISTS AND ANTAGONISTS
ON CARRAGEENAN-INDUCED INFLAMMATION IN RATS
M. Adami, G. Coruzzi, R. Smits, H. Lim, R. Leurs
The paradigm that the histamine H4 receptor (H4R)
challenged by the recently reported agonist activity of JNJ7777120, the ‘‘standard’’ H4R antagonist, in some settings. This is in line with previous observations from our lab, which showed anti-inflammatory effects of the H4R agonists VUF8430 and VUF10460 on carrageenan-induced paw edema in the rat.
In the present study, several H4R ligands were charac-terized for their H4R activity (agonism, partial agonism, neutral antagonism) using the rat GTPcS binding assay and tested in vivo against carrageenan-induced paw edema in the rat.
Among the compounds tested, VUF8430 behaved as a full agonist (a = 0.92 ± 0.02) in the GTPcS assay and induced at 100 mg/kg subcutaneously (sc) a 92 % inhibi-tion of paw edema; JNJ7777120, VUF10460 and clobenpropit behaved as partial agonists (a = 0.42 ± 0.05, 0.13 ± 0.07 and 0.21 ± 0.02, respectively) and induced 77.02, 65.58 and 66.73 % inhibition of paw edema, respectively. VUF10519 and VUF10497 were neutral antagonists in the GTPcS assay (a = 0) and induced 38.18 and 32.36 % inhibition of paw edema, respectively, at 2 h. In conclusion, present data suggest that the functional activity of H4R ligands in rat assays in vivo does not correlate with their functional behaviour in vitro. Surpris-ingly, H4R agonists were more effective than H4R antagonists as inhibitors of carrageenan-induced edema.
Department of Human Anatomy, Pharmacology and Forensic Medicine, Section of Pharmacology, University of Parma, 43125 Parma, Italy. E-mail: maristella.adami@ unipr.it
COMPARATIVE mRNA EXPRESSION PROFILING IN NON-STIMULATED VERSUS H4R STIMULATED
HUMAN LYMPHOCYTES
S. Mommert, D. Pischke, O. Dittrich-Breiholz, M. Kracht, H. Stark, R. Gutzmer, T. Werfel
The histamine H4 receptor (H4R) contributes to the
regu-lation of several major immune functions. In antigen presenting cells, the down-regulation of pro-inflammatory cytokines and chemokines has been observed. In contrast, regarding human lymphocytes, in particular CD4 + T cells, it has been shown that the H4R is up-regulated in Th2
polarized T cells and its activation leads to an increase in the expression of the pro-inflammatory cytokine IL-31. In human Th17 cells stimulation with either histamine or H4R
agonists enhances the production of IL-17.
In order to obtain a more comprehensive insight into the cellular activities of human lymphocytes in response to H4R stimulation, we performed microarray-based mRNA
expression profiling, using the Whole Human Genome Oligo Microarray (G4845A, ID 026652, Agilent Technol-ogies) which contained 44495 oligonucleotide probes covering roughly 27390 human transcripts. For this pur-pose we treated human Th2 polarized T cells, CD8 + T cells and natural killer cells (NK cells) with the selective H4R agonist ST-1006 and compared the mRNA expression
profiles with non stimulated control cells of the same origin.
We detected 13 genes which were differentially regu-lated in the Th2 cell group, 79 genes in the CD8 + T cell group and 17 genes in the NK cell group. Interestingly, gene expression profiling showed no overlap of genes induced via H4R in these three different lymphocyte
sub-types. Regarding Th2 cells, H4R stimulation had no effects
on the expression of those cytokines, chemokines and transcription factors which characterise the classical Th2 phenotype. For CD8 + T cells and NK cells microarray analysis revealed the up-regulation of particular chemokine receptors and chemokines.
Selected targets which were identified by this study will be investigated more thoroughly by qRT-PCR and protein analysis to further clarify the potential of targeting the H4R
for therapeutic benefit in a wide range of immune disorders.
Division of Immunodermatology and Allergy Research, Department of Dermatology and Allergy, Hannover Med-ical School, Carl-Neuberg-Str.1, D-30625 Hannover, Germany. E-mail: Mommert.Susanne@mh-hannover.de
BLOOD HISTAMINE LEVELS AND SKIN H4R
EXPRESSION IN PATIENTS WITH CHRONIC SPONTAEOUS URTICARIA
M.P. Makris, N.L. Lethbridge, E. Zampeli, X.S. Aggelides, P.L. Chazot, E. Tiligada
Chronic spontaneous urticaria (CU) is a common skin disorder characterized by histamine (HA) release from activated skin mast cells and basophils. An underlying cause remains elusive and symptomatic therapies often lack efficacy, thus urging the need for the identification of more beneficial therapeutic strategies. Considering the recent concept of the immunomodulatory role of the HA H4 receptor (H4R), this study aimed to assess the
rela-tionship of blood HA levels and skin H4R expression to the
response of CU patients to standard therapy. Peripheral blood samples were collected from adult male (n = 3) and female (n = 10) CU patients of 45 ± 12 years of age, unresponsive to anti-H1R drugs (group A) and with full
(group B). Whole blood and serum HA levels were deter-mined fluorometrically and presented as ng/ml of blood. H4R expression was detected immunohistochemically on
puncture biopsies (4 mm) from the urticarial lesions. Whole blood HA levels were significantly higher (p \ 0.01) in patients with refractory CU (30.7 ± 12.3, n = 9) compared to responsive subjects (13.3 ± 2.3, n = 4). In contrast, serum HA levels were comparable in the two groups, being 7.7 ± 0.8 and 9.1 ± 1.3 for group A and B, respectively. Interestingly, a significant correlation (r \ 0.05) between whole blood HA levels and monocyte counts was observed in group A samples. Prominent anti-hH4R immunoreactivity was detected in the skin biopsies,
with no apparent change in total skin sample extracts. However, representative samples from the two groups showed differential anti-hH4R expression profiles. These
data provide the first evidence linking whole blood HA levels and monocyte counts in refractory CU. The differ-ential H4R expression profile in skin biopsies from
responsive and unresponsive patients points to a probable role of the H4R in CU that deserves further consideration.
This work was part of FP7 EU COST Action BM0806 Allergy Unit Attikon Hospital, University of Athens Medical School, 12462 Athens, Greece. E-mail: aityliga@ med.uoa.gr
LIGAND-DIRECTED H4R SIGNALLING: A STEP
FORWARD TO OPTIMIZED H4R DRUGS?
S. Nijmeijer, E.M. Rosethorne, H.F. Vischer, S.J. Charlton, R. Leurs
G protein-coupled receptors (GPCRs) acquired their name from the ability to activate G proteins to induce intracel-lular signalling. However, not only G proteins transduce GPCR activation, also other proteins such as b-arrestins can function as signalling scaffolds. Moreover, GPCR ligands do not always activate G protein- and b-arrestin-mediated signalling to an equal amount. This phenomenon is known as ligand-directed signalling and has been observed for various GPCRs.
The histamine H4R was previously shown to couple to
Gai proteins, but has recently been added to the list of
GPCRs that display ligand-directed signalling. The well-known antagonist JNJ 7777120 was surprisingly identified as a partial agonist in a PTX-insensitive PathHunter b-arrestin recruitment assay. In addition, JNJ 7777120 induced ERK phosphorylation in a time frame typical for b-arrestin-mediated signalling.
These observations led us to re-evaluate a variety of H4R compound classes and to investigate their ability to
induce PTX-sensitive CRE activity versus PTX-insensitive b-arrestin recruitment. We have identified compounds with a strong bias towards one of the tested pathways. The newly identified biased H4R compounds will be useful
pharmacological tools to study the functional consequences of biased H4R signalling in future research.
VU University Amsterdam, De Boelelaan 1083, 1081HV Amsterdam, The Netherlands. E-mail: r.leurs@vu.nl
DIFFERENTIAL BINDING KINETICS OF HISTAMINE H4RECEPTOR LIGANDS:
FROM QUICK LEAVERS TO LONG-STAY BINDERS
H.D. Lim, S. Nijmeijer, H.F. Vischer, C. de Graaf, R.A. Smits, I.J.P. de Esch, R. Leurs
The histamine H4receptor is a G protein-coupled receptor
associated with various inflammatory diseases such as allergic asthma, rheumatoid arthritis and pruritus. For this reason, H4 receptor antagonists currently receive
consid-erable attention as potential therapeutics. In our search for new H4ligands, several low affinity fragments were
opti-mized to new high affinity H4 receptor antagonists.
Interestingly, study of the binding kinetics at the human H4
receptor showed very different dissociative half-lives for several compounds. This could potentially explain part of the in vivo effectiveness of e.g. the reference antagonist JNJ 7777120. In conclusion, this study of H4 ligands
indicates important differences in H4 receptor binding
kinetics, which could ultimately be responsible for differ-ences in in vivo activities.
VU University Amsterdam, De Boelelaan 1083, 1081HV Amsterdam, The Netherlands. E-mail: r.leurs@vu.nl
DERIVATIVES OF 2-AMINO-1,3,5-TRIAZINE
AS NEW HISTAMINE H4RECEPTOR LIGANDS
K. Kamin´ska, M. Wie˛cek, T. Kottke, S. Schwed, R. Seifert, H. Stark, J. Handzlik, K. Kiec´-Kononowicz
Histamine—the biogenic monoamine—fulfills important physiological functions through four histamine receptor subtypes (H1-H4). The youngest member of this receptor
family is the H4receptor (H4R) which was discovered at
the turn of 2000 and 2001 year independently by several research groups. Because of H4R expression mainly in cells
and tissues of immune system (monocytes, mast cells, dendritic cells, eosinophils, basophils, T-leukocytes) its
role in inflammatory and (auto)immunological processes and disorders was suggested. Positive effects were observed in treatment of animal models of some diseases (e.g. allergic rhinitis, asthma, pruritus or pain) in the presence of H4R antagonists/inverse agonists.
Extending our SAR investigation in the group of 1,3,5-triazine derivatives—based on previous results and litera-ture data we searched for new potent and selective H4R
ligands in the group of (4-methylpiperazin-1-yl)-1,3,5-triazin-2-amines by changing aryl substituent in 6-position to heteroaryl ring system, e.g. (un)substituted thiophene, benzothiophene, pyridine or imidazopyridine.
The compounds were obtained by the direct reaction of appropriate carboxylic esters with guanidine derivative. In silico predictions of toxicity and drug-likeness by newly obtained compounds were also carried out. Compounds were evaluated for their affinity at H4R with radioligand
binding assays on transiently expressing recombinant human H4R in the Sf9 insect cells using [3H] histamine as
radioligand.
As one of the most promising substitution pattern the thienyl derivatives were characterized with significant affinity to H4 receptor, e.g.
4-(3-chlorothienyl-2-yl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine showed a Ki
value of 137 nM.
Kindly supported by the Polish Ministry of Science and Higher Education Grants No: 594/N-COST/2009/0 and by the FP7 EU COST Action BM0806.
Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Medyczna 9, 30-688 Krako´w, Poland. E-mail: kate.anna.kaminska@gmail. com
MICROSATELLITE REPEAT EXPANSIONS
IN HUMAN HISTAMINE4RECEPTOR GENE
S. Micallef, E. Quinn, D. Morris, A. Sasse
Microsatellite repeat expansions have been implicated in a number of neurological disorders in humans, e.g., Fragile X syndrome, Huntington’s disease and Kennedy’s disease. Both loss-of-function and gain-of-function mechanisms are recognised from expanded trinucleotide repeats, and such microsatellites can be located in both coding and non-coding regions of the gene. This study targets genetic polymorphism in the promoter region of the human hista-mine4receptor (hH4R), which contains variable number of
tandem repeat polymorphisms (VNTR). This VNTR is a CAA triplet repeat expansion and ranges from 10 to 19 repeats (10R and 19R, respectively). In preliminary unpublished data it was indicated that the trinucleotide
repeats 10R, 13R, 14R and 15R, are more common in schizophrenia patients. The aim of this study was to investigate the association of this genetic variation to schizophrenia and to assess promoter activity resulting from this polymorphism. Linkage disequilibrium (LD) analysis was carried out on HapMap samples between the VNTR in the promoter region and known Single Nucleo-tide Polymorphisms (SNPs) at this locus. The SNPs at hH4R were tested for association with schizophrenia as part
of genome wide association studies (GWAS) conducted by the International Schizophrenia Consortium (ISC). Geno-typing showed that the 13R allele is in high LD with rs17797945, while the 10 microsatellite is in high LD with rs628764 and rs615283. The SNP rs17797945 is weakly associated with schizophrenia in the ISC GWAS (P 0.007876). The promoter region including the variations in VNTR have been cloned and are currently being subcloned in a pGL3 promoter-less vector for assessment of promoter activity. This assay will indicate the functional impact which this length polymorphism has on gene expression.
The research work disclosed in this publication is fun-ded by a STEPS Fellowship (Malta). Supported by European COST Action BM0806 ‘Recent Advances in Histamine Receptor H4R Research’.
Trinity College Institute of Neuroscience and School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Ireland. E-mail: sassea@tcd.i.e
SILENCING HUMAN H4R GENE BY NOVEL
TRIPLEX-FORMING MOLECULE
M. Falah, M. Azab, S. Shdafnay, A. Michaeli, A. Rayan
Over the past two decades, there has been considerable interest in developing novel chemicals that can specifically silence a gene and inhibit its expression. Any approach that can specifically and efficiently suppress the expression of genes is recognized to be highly valuable. Two approaches that have received much attention are antisense oligonu-cleotides and siRNA. Both approaches target the RNA product of the expressed gene and in due course result in destruction of this target. An alternative approach that directly targets and inhibits gene expression utilizes tri-plex-forming molecules (TFMs). One advantage of this strategy is the relatively few DNA molecules that need to be targeted (usually 1 or 2 per cell), allowing for dosage reductions that lower toxicity for patients. This provides a distinct advantage over targeting RNA molecules, which are continuously produced when the gene is transcribed, requiring the antisense oligonucleotide or siRNA to be constantly present in the cell. Targeting DNA directly also
prevents biofeedback loops from becoming effective drug resistance mechanisms.
Based on our proprietary bioinformatics tools, we have designed a highly specific molecule that tightly binds a selected DNA sequence along the hH4R gene, stopping its
RNA transcription. This novel synthetic molecule which targets the hH4R gene (hH4R-TFM) is highly selective and
designed not to interact with non-targeted sequences along the human genome. Our TFMs are chemically engineered to bind to the major groove of the targeted DNA fragment. Our molecules interact with the major groove by forming a stable Hoogsteen interaction. This mode of binding is sig-nificantly different from those of antisense oligonucleotides or siRNAs which bind nucleic acids via base-pairing.
The H4R-TFM developed bound efficiently with its DNA
target at physiological temperature and pH, prevented ampli-fication in RT-PCR and, therefore, can now be potentially used to disrupt in vitro transcription and prevent replication of DNA containing a binding site for the hH4R-TFM.
Al-Qasemi Academic College, Baka El-Garbiah 30100, Israel. a_rayan@qsm.ac.il.
THE HISTAMINE H4RECEPTOR
IS OVEREXPRESSED IN THE KIDNEY OF DIABETIC RATS
A.C. Rosa, C. Grange, M.A Katebe, E. Benetti, M. Collino, G. Miglio, G. Camussi, P.L. Chazot, R. Fantozzi
Histamine is known to be synthesized in the glomeruli, to increase second messenger levels in isolated glomeruli and to influence renal hemodynamics, including microcircula-tion. Previous studies demonstrated the glomerular expression of histamine H1receptors (H1R) and H2R, while
the most recently discovered H3R and H4R were poorly
investigated. The aim of this study was to evaluate the H4R
expression in the rat kidney. As renal histamine concen-tration was found to be significantly increased in streptozotocin (STZ)-treated rats, a further aim was to evaluate whether the kidney H4R density varies in diabetic
rats. Insulinopenic diabetes was induced in 12 out of 24 8-week-old male Wistar rats by a single i.v. injection of STZ, and animals were sacrificed 6 weeks later. Serum and urinary evaluation was performed to assess glycaemia, creatinine clearance and proteinuria. The kidneys were collected and processed for PCR or immunohistochemistry analyses. Diabetic rats (glycaemia [300 mg/dl) had a higher creatinine clearance and proteinuria than nondia-betic animals. The H4R mRNA expression in control rats
was at a very low level. Consistently, H4R was
undetect-able by immunohistochemistry using a well validated
anti-H4R antibody. In contrast, in diabetic rats H4R was
pro-foundly upregulated.
Immunohistochemistry experiments revealed a higher immune-positivity in the cortex and medulla in comparison to the papilla. To ascertain the cellular localization of H4R,
colocalization experiments were performed. When H4R
immunostaining was compared to that of Tamm-Horsfall gly-coprotein, a close overlap in expression topology was observed. In conclusion, our results indicated that H4R is
expres-sed in the kidney of healthy rats, although at a very low level, and it is upregulated in diabetic male rats. This receptor is expressed by resident renal cells of the thick ascending limb of the loop of Henle´, thus suggesting a possible role in modulating the transmembrane soluble transport processes.
Funded by Royal College of Anaesthesia/BJA and COST Action BM0806; Dr Rosa performed a successful STSM in the laboratory of Dr Chazot (UK)
Dipartimento di Scienza e Tecnologia del Farmaco, Torino, Italy. E-mail: ariannacarolina.rosa@unito.it
THERAPEUTIC POTENTIAL OF H4R AGONISTS
IN AN EXPERIMENTAL MODEL OF HUMAN BREAST CANCER
D. Martinel Lamas, M. Croci, L. Sambuco, E. Carabajal, N. Massari, R.M. Bergoc, E.S. Rivera, V.A. Medina
We have previously reported the presence of the histamine H4
receptor (H4R) in benign and malignant lesions and cell lines
derived from the human mammary gland. The H4R is one of
the main subtypes responsible for the histamine-induced responses in MDA-MB-231 breast cancer cells. The aim of the present work was to evaluate the effects of H4R ligands on the
survival, tumour growth rate, metastatic capacity and molecular pattern of expression of antigens related with the proliferative and apoptotic potential in a breast cancer experimental model. For that purpose, we established xeno-grafted tumours of the highly invasive human breast cancer cell line MDA-MB-231 in immune deficient nude mice. We employed the following H4R agonists: histamine (1 and 5 mg/
Kg, sc), clozapine (1 mg/Kg, sc), and the experimental com-pound JNJ28610244 (10 mg/Kg, sc).
Results indicate that developed tumours were highly undifferentiated and that the H4R was the major
hista-mine receptor expressed. They also exhibited high levels of histidine decarboxylase, histamine content and proliferation marker (PCNA) while displaying low levels of apoptosis. Mice of the untreated group displayed a median survival of 60 days, and a tumour doubling time exponential growth of 7.4 ± 0.6 days. A significant
decrease in tumour growth evidenced by an augmentation of the tumour doubling time was observed in H4R agonist
groups (13.1 ± 1.2, P \ 0.01 in histamine group; 15.1 ± 1.1, P \ 0.001 in clozapine group; 10.8 ± 0.7, P \ 0.01 in JNJ28610244 group). The effect was associ-ated with a decrease in the expression levels of the H4R
(32.8 vs. 6.6 % and 3.0 in clozapine and JNJ28610244 groups, respectively, P \ 0.05) and of the PCNA (83.1 vs. 63.2 %, 64.2, 70.5 % in histamine, clozapine and JNJ28610244 groups, respectively, P \ 0.05). Histamine treatment significantly increased median survival (78 days; ***P \ 0.0005, Log rank test and Gehan-Breslow-Wilco-xon test). We conclude that histamine through the H4R
exhibits a crucial role in tumour progression. Therefore, H4R ligands offer novel therapeutic potential as adjuvants
for breast cancer treatment.
We thank Dr. Nicholas Carruthers from Johnson & Johnson Pharmaceutical Research & Development for the JNJ28610244 compound.
Laboratorio de Radioiso´topos, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, 1113, Buenos Aires, Argentina. E-mail: vmedina@ffyb.uba.ar
ESTABLISHMENT OF A HISTAMINE METHODS AND TOOLS DATABASE
H.G. Schwelberger
COST Action BM0806 Recent Advances in Histamine Receptor H4R Research brings together scientist and
industrial partners from many different disciplines, who
provide enormous expertise in their respective field. In order to make this expertise transparent and usable for all action members and for histamine researchers in general, one goal of Working Group 1 is to establish and run an internet database covering all methods and tools available at participating institutions.
In the first phase to build this database, a simple ques-tionnaire was used to collect, evaluate and combine information on available methods and tools from individual participants. This methods and tools overview will serve as a contents and reference point for users. In the second phase, detailed protocols and descriptions of tools are collected from individual contributors and made available for users in a common, easy to use format. In the third phase, the database will be constantly updated with new information and errors will be corrected based on user feedback.
In its final version, this histamine methods and tools database should provide a comprehensive collection of all available methods (techniques, assay systems, cell systems, animal models, patient based studies), include ready-to-use protocols, give contact information for method based inquiries (technical help), inform about source of critical tools (antibodies, reagents, compounds), and finally iden-tify areas where new methods, techniques or tools should be developed.
This work was supported by COST Action BM0806. Medical University Innsbruck, Department of Visceral, Transplant and Thoracic Surgery, Molecular Biology Laboratory, Scho¨pfstrasse 41, A-6020 Innsbruck, Austria. E-mail: hubert.schwelberger@i-med.ac.at
UNDERSTANDING HOW HISTAMINE INDUCES NEURON DIFFERENTIATION
G. Rodrı´guez, I. Velasco, G. Garcı´a-Lo´pez,
H. Flores-Herrera, N.F. Dı´az, A. Molina-Herna´ndez
Histamine is a neurotransmitter in the central nervous system. This amine is one of the first neurotransmitters to appear during development and it reaches its maximum concentration at the same time as neuron differentiation peak. This suggests that histamine has an important role in neurogenesis. We have previously shown that hista-mine is able to increase neuronal differentiation of neural stem cells in vitro, by activating the histamine H1
receptor. In this study, we start to explore the mechanism involved in neuronal differentiation stimulated by hista-mine. We used rat neural stem/progenitor cells from embryonic day 14 in passage two. We performed two protocols of histamine treatments; one when it was present during both cell proliferation and differentiation and the other where histamine was only present during cell differentiation.
Immunocytochemistries were performed to evaluate cell division and neuron differentiation. RT-PCR or qRT-PCR were used to explore the expression of some important transcriptional factors involved on cell com-mitment. Results indicate that histamine is required during cell proliferation in order to increase neuron dif-ferentiation, probably by increasing neuron commitment, since histamine is able to increase asymmetric cell divi-sion during the proliferative phase as well as the expression of FGF-receptor type 1, Neurogenina 1 and Prospero 1 expression. We proposed that the increase on deep layer cortical neurones, measured by an increase on FOXP2-positive cells, due to histamine is related to: (a) an increase on asymmetric cell division and (b) an increase on FGF-receptor 1, Prospero 1 and Neurogenin 1 expression. These effects might be related to the acti-vation of histamine H1 receptor, since the presence of
chlorpheniramine prevented the increase on FGF-receptor 1 expression.
Departamento de Biologı´a Celular. Instituto Nacional de Perinatologı´a ‘‘Isidro Espinosa de los Reyes’’, Torre de Investigacio´n. Montes Urales 800, Delegacio´n Miguel Hidalgo. Colonia Lomas de Virreyes, CP 11000. Me´xico City, Mexico. E-mail: anayansimolina@gmail.com.mx
INHIBITION OF DEPOLARIZATION-EVOKED [3H]-DOPAMINE RELEASE BY THE ACTIVATION
OF THE HUMAN HISTAMINE H3RECEPTORS
OF 445 AND 365 AMINO ACIDS EXPRESSED IN HUMAN NEUROBLASTOMA SH-SY5Y CELLS G. Nieto-Alamilla, J. Escamilla-Sa´nchez,
R. Gonza´lez-Pantoja, J.-A. Arias-Montan˜o
The histamine H3 receptor (H3R) is mainly located on
nerve terminals and controls release of histamine and other transmitters, including dopamine. The alternative splicing of the human H3R gene produces several receptor isoforms,
of which variants of 365 and 445 amino acids (hH3R365and
hH3R445) are expressed in brain. The aim of this work was
to determine, by using a cellular model, whether these two isoforms regulate in a different manner neurotransmitter release.
Two sub-lines of human neuroblastoma SH-SY5Y cells stably expressing either isoform (hH3R365 or hH3R445)
were generated with expression levels of 583 ± 151 and 805 ± 175 fmol/mg protein and Kd values for [3 H]-N-methyl-histamine of 0.90 ± 0.13 and 0.86 ± 0.12 nM, respectively. There were no significant differences in the affinities of both isoforms for the H3R ligands histamine
(pKi values 8.50 ± 0.10 and 8.55 ± 0.07), R-a-methyl-histamine (RAMH, 9.45 ± 0.06 and 9.63 ± 0.06) or clobenpropit (8.70 ± 0.09 and 8.95 ± 0.12).
The H3R agonist RAMH was more efficacious to
inhibit forskolin-induced cAMP accumulation in the SH-SY5Y-hH3R445cell line (-70.6 ± 5.2 % vs. -38.4 ± 5.4
% for the SH-SY5Y-hH3R365 cells), without significant
difference in the pIC50 estimates (8.77 ± 0.17 vs. 8.42 ± 0.14).
Neurotransmitter release was assayed in cells differenti-ated by treatment for 7 days with 12-O-tetradecanoyl-phorbol-13-acetate, TPA (20 nM), and retinoic acid (10 lM). The release of [3H]-dopamine induced by depo-larization with high K+(100 mM) was significantly reduced by RAMH (100 nM), but the effect was significantly larger in the SH-SY5Y-hH3R445cell line (-30.9 ± 4.2 %) than in the
cells expressing the hH3R365isoform (-15.1 ± 4.8 %).
Our results indicate that in accord with previous data for other signaling pathways, the hH3R445 isoform is more
efficacious in inhibiting depolarization-evoked neuro-transmitter release.
